Mark A. Carver

Directed evolution of amidase in Methylophilus methylotrophus; purification and properties of amidases from wild-type and mutant strains

Summary: The obligately methylotrophic bacterium Methylophilusmethylotrophus hydrolyses acetamide and acrylamide using a cytoplasmic amidase. In previous work, continuous culture was used to isolate spontaneous mutants which overexpressed either the wild-type amidase (strain MM6) or a mutant amidase with an apparently higher K cat (strain MM8). We now report that NTG mutagenesis of strain MM8 followed by acrylamide-limited growth at low dilution rate (D 0·025 h-1; 37 °C) led to the selection of a strain which continued to overexpress the amidase, but which exhibited an unexpectedly low amidase activity and a greatly decreased K m for acrylamide (strain MM15). Amidases from the wild-type and mutant strains were purified and shown to be homotetramers (subunit M r 38000, pI 4·1). The N-terminal amino acid sequence of the wild-type enzyme was 90% homologous with the aliphatic amidase from Pseudomonas aeruginosa, and Southern blotting using an oligonucleotide probe for this region showed that overexpression of the enzyme in the mutant strains was not due to gene amplification. Compared with the wild-type and MM6 enzymes, the MM8 enzyme exhibited a threefold higher K m and a slightly lower K m for acrylamide, whereas the MM15 enzyme exhibited a similar K cat and an eightfold lower K m for acrylamide. The MM15 enzyme also reacted more extensively with the thiol group reagent DTNB, had a significantly lower sedimentation coefficient and exhibited a more relaxed substrate specificity, all of which were compatible with a looser tetrameric structure. It was also much more susceptible than the other three enzymes to inactivation by high temperature or by freezing and thawing (MM15“MM8>MM6/wild-type), both of which variably dissociated the enzyme into inactive dimers and monomers. The amidase activity of strain MM15 was almost 15-fold higher following growth at 25 °C than at 37 °C, since at this lower temperature the enzyme exhibited a similar K cat to the MM8 enzyme and was not significantly dissociated. However, as strain MM15 readily outgrew the organism from which it was derived (strain MM8) during acrylamide-limited continuous culture at 37 °C, it is clear that under these conditions a low K m was a greater selective advantage than a high K cat.

The terminal respiratory chain of the methylotrophic bacterium Methylophilus methylotrophus

Cytochrome oxidase o has been isolated from the obligately aerobic, methylotrophic bacterium Methylophilus methylotrophus in the form of a cytochrome c L‐o complex. The latter is comprised of cytochrome c L (M r 21 000) and cytochrome o (M r 29 000) in a 1–2:1 ratio, possibly in association with one or more minor polypeptides; the complex exhibits a high ascorbate‐TMPD oxidase activity which is inhibited non‐competitively by cyanide (K i ≈ 2 μM). In contrast, the oxidation of methanol by whole cells is inhibited uncompetitively by cyanide (K i≈4 μM), thus indicating the involvement in methanol oxidation of cytochrome oxidase aa 3 rather than o.

Physiology of amidase production by Methylophilus methylotrophus: isolation of hyperactive strains using continuous culture

Summary: The obligately methylotrophic bacterium Methylophilus methylotrophus hydrolyses aliphatic amides to ammonia and aliphatic acid using a cytoplasmic amidase. Physiological regulation of amidase activity was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. The results showed that synthesis of the enzyme was induced by various amides (acrylamide > acetamide) and repressed by ammonia. Growth of the wild-type organism in acetamide-limited continuous culture at very low dilution rate (D 0·025 h−1) led to the selection of a hyperactive strain (MM6), the subsequent growth of which under acrylamide limitation led to the selection of another strain (MM8) which showed even higher activity. The amidase activities of strains MM6 and MM8 were respectively approximately four and twelve times higher than that of the wild-type organism following growth under similar conditions, whereas the concentrations of the enzyme as determined by SDS-PAGE and scanning densitometry were approximately four times higher than the wild-type organism in both strains. The amidase in strain MM8 exhibited a K m for acrylamide that was approximately one-third lower than that of the wild-type organism or of strain MM6. It is concluded that the hyperactivity of strain MM6 was due predominantly to the production of more wild-type enzyme, whilst the hyperactivity of strain MM8 was due to the production of approximately the same amount of enzyme as strain MM6 (up to 25% of the total cell protein depending on the nature of the limiting amide) but with a substantially enhanced catalytic activity (K cat). These changes were apparently the result of spontaneous mutations that occurred in response to growth at extremely low amide concentrations, giving the novel strains a strong selective advantage under these conditions (possibly by enhancing the rate of diffusion of amide into the cell).

The role ofc-type cytochromes in the terminal respiratory chain of the methylotrophic bacteriumMethylophilus methylotrophus

Whole cells of the methylotrophic bacteriumMethylophilus methylotrophus cultured under methanol-limited conditions contain approximately equal amounts of two majorc-type cytochromes,cH andcL. Virtually all of the cytochromecH, and over one-third of the cytochromecL, are loosely attached to the periplasmic surface of the respiratory membrane whence they can be released by sonication or by washing cells in ethylenediaminetetraacetate (EDTA). The latter causes inhibition of methanol oxidase activity and stimulation of ascorbate-N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) oxidase activity, neither of which effects are reversible by divalent metal ions. Kinetic analyses indicate that ascorbate-TMPD is oxidised via two routes, viz. a slow low-affinity pathway involving loosely membrane-boundc-type cytochromes plus cytochrome oxidaseaa3, and a faster higher-affinity pathway involving the firmly membrane-bound cytochrome oxidasecLo complex; the former route predominates in the presence of divalent metal ions, and the latter route after exposure to EDTA. These and other results are discussed in terms of the spatial organisation of the terminal respiratory chain, and of the role ofc-type cytochromes in the oxidation of methanol and ascorbate-TMPD.

Evidence that the low-affinity K+ transport system of Rhodobacter capsulatus is a uniporter. The effects of ammonium on the transporter

Comparison of the rate of accumulation of 86Rb+ by intact cells of Rhodobacter capsulatus during short periods of illumination with the Rb+-dependent membrane ionic current measured by electrochromism supports the view that both activities reflect the operation of a low-affinity K+ transport system. In experiments performed under similar conditions the ratio of 86Rb+ uptake to charge uptake was approx. 1.0, suggesting that the transport system operates as a uniporter. The addition of NHinf4sup+to a cell suspension led to an increase in membrane ionic current but failed to inhibit the accumulation of 86Rb+ during illumination. The presence of K+ and NHinf4sup+inhibited the increase in cellular ATP levels at the onset of illumination. This effect was prevented by Cs+. The results are considered within the context of the hypothesis (Golby et al. Eur J Biochem 187: 589–597) that NHinf4sup+can be transported by the K+ carrier and in the context of an alternative hypothesis that NHinf4sup+increases the affinity of the K+ transport system for its natural substrate and for Rb+.